Downstream process for purifying polysaccharides

ABSTRACT

The present invention relates to a novel process for purifying bacterial polysaccharide. It is an efficient and scalable process for removing impurities from  Neisseria meningitidis  serogroup C (Men-C) polysaccharide which is capable of being used as such in a derivatized form or linked to other molecules, for the preparation of vaccines, more particularly conjugate vaccines for  N. meningitidis  infection.

FIELD OF THE INVENTION

The present invention relates to a rapid process for purifying Neisseriameningitidis polysaccharide. More specifically, the present inventionrelates to a process of preparing N. meningitidis serogroup type C(MenC) polysaccharides capable of being used as such, or of beingderivatized or combination to other serogroups to make vaccines for N.meningitidis.

BACKGROUND OF THE INVENTION

Polysaccharides, especially antigenic polysaccharides, are used inpreparation of vaccines. Monovalent, bivalent and poly (multi) valentvaccines containing one, two or more polysaccharides and theirconjugates are available in the market for prevention of certaindiseases or infections caused by various microorganisms such asStreptococcus pneumoniae, Haemophilus influenzae and N. meningitidis andhave proved valuable in preventing the respective diseases to asignificant extent. Surveillance data gathered in the years followingthe introduction of the vaccine Prevenar has clearly demonstrated areduction of invasive pneumococcal disease in US infants as expected.Despite of several studies carried out on these polysaccharides andconjugates, a need for improving yields as well as quality (purity) ofthe polysaccharides always exist in the industry as evidenced by thecontinuing research.

Meningitis is considered as a global threat and the disease burden stillremains a public health priority. The clinical definition of meningitisis the inflammation of the meninges (membrane of the brain). If nottreated, the disease can have fatal consequences. A total of thirteenserogroups of N. meningitidis have been identified, and out of thesethirteen, six serogroups (A, B, C, W135, X and Y) are majorlyaccountable for causing the infections globally. N. meningitidis has awide range of clinical manifestations, ranging from transient mild sorethroat to fatal meningitis or meningococcal septicemia. Meningitis andsepticemia are the most common presentations of the disease.

Evidence collected through numerous research findings defines theimmunogenic aspect of the polysaccharide conjugate vaccine. Furthermore,there are research articles as well as patents describing the productionand purification of the capsular polysaccharide of Men C but none ofthem define a purification process with extremely reduced time lineswhich is robust, scalable, and reproducible.

The production of purified Men C is the foremost requirement for aneffective conjugation with the carrier protein and its development as aconjugate vaccine. The cost for the cultivation and the purification ofMen C is generally high and involves long working hours as it involves aseries of production and purification steps. Improvement in one or moreof the steps of polysaccharide production would bring a significantchange in the overall conjugate vaccine production and consequentlymakes the process relatively cost effective.

However, despite of several studies that have been carried out on thesepolysaccharides, there has always been a need for improving yields aswell as quality/purity of the polysaccharides in order to producevaccines of high quality.

There are a number of patents, which describe the process for thepurification of Men-C polysaccharides. The existing state of the artdescribed in U.S. Pat. No. 7,491,517,B2 for precipitating Men-Cpolysaccharides with CTAB is found to involve overnight incubation at 4°C. Also the removal of contaminants requires the use of costly enzymeProteinase K. In addition to this, the process also requires gelfiltration for the purification of the Men-C polysaccharides. Theoverall procedure requires significant time for purification and alsothe process becomes costly because of the use of enzymes.

Also, the Application No. WO 2011/148382 A1 describes the method ofpreparing pure capsular polysaccharide using aluminium phosphate withalcohol for the purification of capsular polysaccharides of Haemophilusinfluenzae b, N. meningitidis such as serogroups A, C, Y, W-135 andother similar capsular polysaccharides produced form both gram negativeand gram positive microorganisms. The said published prior art disclosesa time of 16-20 hrs for the purification of the polysaccharides.

Another patent EP 0658118B1 describes a method for O-Deacetylation ofGroup C Meningococcal Polysaccharide (GCMP) with an average time of 16hours. This process also requires significant time for purification

Presently, the various methods used for the production and purificationof MenC polysaccharides takes relatively long cultivation andpurification time and also involves the use of costly enzymes. Thoughthese kinds of processes give pure polysaccharides but they willconcurrently increase the cost of production during scale-up.

Furthermore the above disclosed prior arts teach methods which are moreefficient at low temperature thereby requiring a controlled environmentleading to addition of costs in research and production.

OBJECT OF THE INVENTION

Thus the main object of the present invention is to provide a novelprocess for the purification of Men C polysaccharide.

Another object of the invention is to provide a rapid process forpurification of Men C polysaccharide while eliminating the impurities ina very short time by simple, efficient, improved and commerciallyscalable method.

Yet another object of the present invention is to produce high qualitypolysaccharide which meet the relevant WHO specifications.

SUMMARY OF THE INVENTION

The present invention relates to the process for purification of Men Cpolysaccharide. The process describes a novel, rapid, cost effective,and scalable method, wherein Men C polysaccharide is purified withsignificantly reduced time.

The process of the instant invention involves the selection ofimmunologically active bacterial strain, preparing the media for thepropagation of said immunologically active strain and inoculating theselected bacterial strain in glycerol stock culture and incubating it atpredefined temperature for an optimal time period at predefinedrevolutions per minute (rpm). The selected bacterial strain iscultivated on optimized cultivation media in the fermenter and theprocess proceeds by doing the centrifugation of the fermented harvest toclarify the fermentation broth (FB) of cell debris followed byconcentration of the FB by ultrafiltration using molecular weightcut-off membranes.

The ultra-filtered concentrated supernatant or the processed FB is thendeacetylated in presence of high concentration of alkaline solution athigh temperature. The deacetylated crude polysaccharide is subjected tobuffer exchange through Tangential Flow Filtration (TFF). Diafiltereddeacetylated crude polysaccharide is further purified by hydrophobicinteraction chromatography (HIC) followed by diafiltration andconcentration to get the final purified polysaccharide. Of particularrelevance is the method according to the invention which comprises thetreatment of a concentrated extract and/or isolated bacterial cells witha basic solution. In addition to extracting the CPS, the base extractionalso causes deacetylation of N-acetyl groups. The extent of thedeacetylation may be varied by adjusting the reaction conditions. Theextracted CPS are then separated from the cellular components to obtainthe CPS preferably by chromatographic separation.

The process exhibits a number of advantages over prior art, such asproviding a novel and rapid method of preparing Men C polysaccharide.The process is cost effective as it reduces the total number of stepsand requires single chromatographic screening. An additional advantageis that this process is entirely scalable.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 Depicts the process flow for the MenC-PS purification andrecovery.

FIG. 2 Depicts the HPLC Chromatogram of MenC-PS

FIG. 3 Depicts the NMR spectrum of MenC-PS.

DETAILED DESCRIPTION OF THE INVENTION

The invention discloses two steps that have been optimized to enable thepurification of MenC polysaccharide (MenC-PS) in lesser time as shown inFIG. 1.

The strain of bacterial polysaccharide is inoculated in the fermentercontaining appropriate media components required for bacterial growth.After achieving the maximum optical density, in the range of 8 to 10depicting substantial bacterial growth, it is subjected to terminationby adding prerequisite concentration of formaldehyde and the resultantFB is obtained. FB is then centrifuged at high speed to clarify the FBof cell debris followed by diafiltration and concentration usingmolecular weight cut-off membranes.

The diafiltered and concentrated FB having the crude polysaccharide istreated with NaOH at high temperature e.g up to 80° C. fordeacetylation. Concentration and diafiltration of the crudepolysaccharide is performed with polyether sulfone (PES) membrane, withmilliQ water (MQW), followed by Tris HCl buffer pH 7.4±0.1. Afterdiafiltration crude polysaccharide is concentrated and filtered with0.22 μm filter.

Deacetylated crude polysaccharide is further purified by Hydrophobicinteraction chromatographic (HIC) technique.

The separation of two or more components of a mixture based ondifferences in polarity is well known to those skilled in the art. Forexample, using hydrophobic-interaction chromatography, compounds ofrelatively greater hydrophobicity are retained longer on the columnrelative to those compounds that are more hydrophilic. Conversely, usinghydrophilic-interaction chromatography, hydrophilic compounds areretained longer on the column relative to those compounds that are morehydrophobic. Using both methods consecutively allows for the removal ofimpurities that are both less polar and more polar relative to thecompound of interest

The extracted CPS present in the base extraction reagent can beseparated from impurities resulting from the cellular components bychromatography. Non-limiting examples of the chromatographic separationmethods are ion-exchange (cationic or anionic), hydrophilic-interaction,hydrophobic-interaction or gel-permeation chromatography. More preferredis hydrophobic-interaction chromatography (HIC) on phenyl sepharosewhich will remove most of the high molecular weight, uv-activecontaminants from the base extract. Capsular polysaccharide will elutein the beginning, while, the more hydrophobic protein and nucleic acidswill be retained. The preferred method in this invention ishydrophobic-interaction chromatography.

Further concentration and diafiltration of the polysaccharide isperformed to facilitate the removal of proteins and other impurities,and the polysaccharide sample is then washed with MQW.

Consequently sterile filtration is done to obtain the purified Men Cpolysaccharide depicted by FIGS. 2 and 3.

The corroboration of the above employed procedures can be convenientlyunderstood from Table 1 which clearly shows that the purifiedpolysaccharide specifications meet the WHO standard.

TABLE 1 Purified Men-C polysaccharide specifications in accordance withWHO specification are shown below Tests MenC-PS WHO SpecificationsPolysaccharide content 4 mg/ml Actual value Sialic acid content  100%The sialic acid for MenC content not less than 80% of the dry weightEndotoxin <0.06 EU/μg Less than 100 EU/μg Protein % 0.14% Not more than1% Nucleic Acid % 0.42% Not more than 1% Average relative 351 kd Actualvalue from HPLC Molecular Weight Identity Positive Positiveagglutination reaction with specific sera; Defined ¹H- NMR spectrum

Of particular interest is the observation that the time required topurify the Men C polysaccharides is significantly less from thosedisclosed in the prior arts whereas the hallmark of the presentinvention is the novel, rapid and scalable purification process protocolwhich can be completed within 6±1 hours and the PS so produced is incompliance with the WHO set standard.

Analytical Procedures:

Polysaccharide obtained at different steps are constantly monitored andanalyzed for their purity and yield. Different analytical procedures arereported but the preferred ones are as summarized below:

Total sialic acid of MenC-PS is determined by a colorimetric method. Theassay is based on the reaction between sialic acid and resorcinol at100° C. in the presence of hydrochloric acid and copper (II) ions for 30minutes; this leads to the formation of a blue-violet complex whichexhibits a strong absorbance at 564 nm. The total sialic acidconcentration is determined from a calibration curve obtained using aseries of sialic acid standards [(Svennerholm, 1957)].Lipopolysaccharide (LPS) is determined using compact and simpleEndosafe®-PTS™ apparatus. Protein impurity is determined by Lowry'smethod [(Lowry et al., 1951)] using bovine serum albumin as a standardand the absorbance is taken at 750 nm. Nucleic acids (NA) is estimatedat 260 nm and the amount is calculated assuming an absorbance of 1.0A=50 μg/mL [(Frasch, 1990)].

Relative Average molecular size (Mw) of HibPRP is determined usingHigh-performance liquid chromatography (HPLC) (Alliance, Waters). Thecolumns used are PWXL-4000 and PWXL-5000 in series (Tosoh Bioscience).Furthermore, a range of 5 kD to 800 kD Pullulans (Shodex) are used asstandards for MenC-PS. The HPLC is performed using 0.1 M sodium nitratewith a run time of 30 min at a flow rate of 1 ml/min. The identity ofMenC-PS is verified by 1H-NMR spectroscopy. NMR yields a spectrum ofmagnetic sensitive nuclei (e. g. 1H).

The MenC-PS is identified serologically by combining with the referenceantisera against each polysaccharide. As the WHO specifications [WHO,2004] to determine the purity and to characterize the polysaccharide isbased on dry weight basis, the MenC-PS is first lyophilized and thentested. The moisture content is thus subtracted to get the exact dryweight. Moisture content of lyophilized cake is determined by Thermogravimetric Analyzer (TGA) from Perkin Elmer. The analytical results forMenC-PS are given in Table-1 and are in accordance and as specified byWHO.

The said Men C polysaccharide can be used for the preparation ofpolysaccharide-protein conjugate vaccines.

Various aspects of the invention described in details above is nowillustrated with non-limiting examples.

EXAMPLE-1

Polysaccharide Purification Using Anionic Detergent with Tris Buffer andHydrophobic Interaction Chromatography (HIC):

The diafiltered and concentrated FB having the crude polysaccharide isprocessed with anionic detergent, such as, sodium deoxycholate at aconcentration 0.3% to 0.6% (w/v) and incubated at 50° C.-60° C. for 1hour. Subsequently it is cooled to below 40° C. followed byconcentration and diafiltration of the crude polysaccharide with 0.1 m²poly ether sulfone (PES) membrane, with 6-8 volumes of milliQ water(MQW).

Deacetylation is then performed on the crude polysaccharide with 0.5Msodium hydroxide (NaOH) at 50° C. for 10 hours. Subsequently it iscooled to below 40° C. followed by diafiltration and concentration ofthe crude polysaccharide simultaneously with 6-8 volumes of MQW and 20mM Tris HCL buffer using 0.1 m² PES membrane. Afterwards thepolysaccharide is further purified by Hydrophobic InfractionChromatography (HIC), Such as, Phenyl sepharose 6 Fast Flow. Finally,concentration and diafiltration of the purified polysaccharide wasperformed with 0.1 m² PES membrane, with 6 to 8 volumes of MQW.Accordingly, sterile filtration is done with 0.22 μm filter and thefinal purified polysaccharide is stored at −20° C. The total time takento purify the polysaccharide including HIC chromatography using theabove process was 16-18 hours.

EXAMPLE-2

Polysaccharide Purification Using Anionic Detergent with HEPES Buffer(4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid) and HydrophobicInteraction Chromatography (HIC):

The diafiltered and concentrated FB having the crude polysaccharide isprocessed with anionic detergent, for example, sodium deoxycholate at aconcentration 0.3% to 0.6% (w/v) and incubated at 50° C.-60° C. for 1hour. Subsequently it is cooled to below 40° C. followed byconcentration and diafiltration of the crude polysaccharide with 0.1 m²poly ether sulfone (PES) membrane, with 6-8 volumes of MQW.

Deacetylation is then performed on the crude polysaccharide with 0.5Msodium hydroxide (NaOH) at 50° C. for 10 hours. Subsequently it iscooled to below 40° C. followed by diafiltration and concentration ofthe crude polysaccharide simultaneously with 6-8 volumes of MQW and 20mM HEPES containing 3M NaCl buffer using 0.1 m² PES membrane. Afterwardsthe polysaccharide is further purified by Hydrophobic InteractionChromatography (HIC), Such as, Phenyl sepharose 6 Fast Flow.Concentration and diafiltration of the purified polysaccharide wasperformed with 0.1 m² PES membrane, with 6-8 volumes of MQW.Consequently, sterile filtration was done with 0.22 μm filter and thefinal purified polysaccharide is stored at −20° C. The total time takento purify the polysaccharide including HIC chromatography using theabove process was 16-18 hours.

EXAMPLE-3

Polysaccharide Purification Using Sodium Hydroxide with HEPES Buffer((4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid) and HydrophobicInteraction Chromatography (HIC):

The diafiltered and concentrated FB having the crude polysaccharide isdeacetylated with 0.8M NaOH for 6 hours at 80° C. Subsequently it iscooled to below 40° C. followed by diafiltration and concentration ofthe crude polysaccharide simultaneously with 6-8 volumes of MQW and 20mM HEPES containing 3M NaCl buffer using 0.1 m² PES membrane. Afterwardsthe polysaccharide is further purified by Hydrophobic InteractionChromatography (HIC), Such as, Phenyl sepharose 6 Fast Flow.Concentration and diafiltration of the purified polysaccharide wasperformed with 0.1 m² PES membrane, with 6-8 volumes of MQW.Consequently, sterile filtration was done with 0.22 μm filter and thefinal purified polysaccharide is stored at −20° C. The total time takento purify the polysaccharide including HIC chromatography using theabove process was 10-12 hrs. Moreover the polysaccharide is partiallypurified with the above mentioned process. The process may require fewmore steps for complete purification of the polysaccharide and may notbe a cost effective process.

EXAMPLE-4

Polysaccharide Purification Using Sodium Hydroxide with Tris Buffer andHydrophobic Interaction Chromatography (HIC):

The diafiltered and concentrated FB having the crude polysaccharide isdeacetylated with 0.8M NaOH for 6 hours at 80° C. Subsequently it iscooled to below 40° C. followed by diafiltration and concentration ofthe crude polysaccharide is done simultaneously with 6-8 vols of MQW and20 mM Tris HCL buffer using 0.1 m² PES membrane. Afterwards thepolysaccharide is further purified by Hydrophobic InfractionChromatography (HIC), Such as, Phenyl sepharose 6 Fast Flow.Concentration and diafiltration of the purified polysaccharide wasperformed with 0.1 m² PES membrane, with 6 to 8 volumes of MQW.Accordingly, sterile filtration is done with 0.22 μm filter and thefinal purified polysaccharide is stored at −20° C. The total time takento purify the polysaccharide including HIC chromatography using theabove process is 10-12 hours. Moreover the polysaccharide is partiallypurified with the above mentioned process. The process may require fewmore steps for complete purification of the polysaccharide and may notbe a cost effective process.

EXAMPLE-5

Polysaccharide Purification Using Sodium Hydroxide with Tris Buffer andHydrophobic Interaction Chromatography (HIC):

The diafiltered and concentrated FB having the crude polysaccharide wasdeacetylated with 1M NaOH for 2 hrs at 75±5° C. The deacetylatedpolysaccharide was then cooled to a temperature below 40° C. Aftercooling, the concentration and diafiltration of the crude deacetylatedpolysaccharide was performed through 100 KDa PES membrane (0.1 m²) with20 to 25 volumes of MQW, followed by 8-10 volumes of 20 mM Tris HClbuffer (pH 7.4±0.1). Consequently sterile filtration was done with 0.22μm PES membrane (0.1 m²).

Daifiltered deacetylated polysaccharide was further purified byhydrophobic interaction chromatrography (HIC) through XK-16 columnpacked with phenyl sepharose 6 fast Flow (FF) using chromatographysystem. The column was equilibrated with 20 mM Tris HCl buffer pH7.4±0.1 containing 20% Ammonium Sulphate. The material was loaded at 60cm/hr. and the flow through containing the purified polysaccharide wascollected. Finally column was regenerated with 20 mM Tris HCl buffer pH7.4±0 and stored in 20% ethanol for further use. Concentration anddiafiltration of the purified polysaccharide was performed with 100 KDaPES membrane (0.1 m²), with 6-8 volumes of MQW and sterile filtration isdone with 0.22 μm filter and the final purified polysaccharide is storedat −20° C. The total time taken to purify the polysaccharide is 6±1hours and the PS qualifies to the WHO specifications.

We Claim:
 1. A process for purifying Men C-Polysaccharide, wherein saidprocess comprises the steps of: (a) centrifugation of the fermentedharvest to clarify the fermented broth; (b) concentrating the fermentedsupernatant by ultrafiltration; (c) deacetylation and incubating saidconcentrated supernatant of step (b) at a temperature ranging from 70°C. to 80° C.; (d) collecting the supernatant of step (c) and subjectingto diafiltration and concentration; (e) purification of diafilteredsupernatant of step (d) by chromatography; (f) diafiltration andconcentration to obtain purified polysaccharides; and (g) sterilefiltration of said purified polysaccharides, wherein the saidpurification process is completed within 6±1 hours and wherein thepurification process is completed without the use of a detergent.
 2. Theprocess as claimed in claim 1 wherein said step of centrifugation of thefermented harvest is carried at 4500×g to 5000×g.
 3. The process asclaimed in claim 1 wherein said step of deacetylation is carried withNaOH solution at a concentration which ranges from 1 to 1.5 M.
 4. Theprocess as claimed in claim 1 wherein the incubation in thedeacetylation step is performed with a total incubation time rangingfrom 1 hour 30 mins to 2 hours 30 min.
 5. The process as claimed inclaim 1 wherein the diafiltration in the collecting step after thedeacetylation step is carried out 20 to 25 times with Milli-Q waterfollowed by 8 to 10 times with 25 mM Tris HCl buffer.
 6. The process asclaimed in claim 1 wherein said chromatography used in the purificationstep is hydrophobic interaction chromatography (HIC) Phenyl sepharose 6fast flow.
 7. The process as claimed in claim 1 wherein thediafiltration and concentration to obtain purified polysaccharides stepis carried with 100 KDa PES membrane (0.1 m²), with 6 to 8 volumes ofMilli-Q water.